Bovine Herpesvirus 1 (BHV-1) is a significant pathogen of cattle. Infection with BHV-1 in cattle has also been termed "Infectious Bovine Rhinotracheitis" (IBR); "Infectious Pustular Vulvovaginitis" (IPV); and "Infectious Balanoposthitis" (IBP) (Pastoret et al., 1984, p.211-228 In: Latent herpes virus infections in veterinary medicine, (eds. Wittman, Gaskell, and Rizha), Martinus Nijhoff Publishers; and Straub et al., 1990, In: Virus infections in ruminants, (eds. Dinter and Morein), Elsevier Publishers). BHV-1 can cause abortions and respiratory, genital, and central nervous system diseases. Apparently, the mode of transmission is the cause of the various disease states, rather than variations in the virus serotype. BHV-1 is highly contagious and high titers of the virus can be excreted from the portal of entry, i.e. the virus can be found in saliva, nasal secretions, ocular discharges, genitourinary discharges, and semen of infected animals, depending on the various disease state. Of particular clinical and economic importance, bovine semen is capable of transmitting the disease to both cows and fetuses. The risk of transmission by semen is increased through the use of artificial insemination, where a single virus laden ejaculate may be diluted and inseminated in many susceptible cows (Drew et al., 1987, Vet. Rec. 121:547-548). It is important to note that the virus can survive in semen storage containers below -65.degree. C., where it may also contaminate virus-free semen. Transmission of the viral agent can occur from an animal showing overt symptoms of disease, as well as during a subclinical infection where the appearance of disease or lesions is absent.
Once an animal is infected, and despite the infected animal mounting an adequate immune response, the virus may remain latent in ganglia and may be re-excreted in the respiratory and/or genital tract. Clinical recurrence can follow from reactivation of the virus from the latent state when an animal is stressed or given corticosteroids. Treatment of the disease is palliative, however, various vaccine formulations are available for use. Whole virus vaccines have limited use since killed vaccines often do not induce an immune response sufficient for providing adequate protection; and live virus vaccines can cause latency, with the possibility of reactivation, and often subsequent undesirable sequelae. BHV-1 polypeptide vaccines are being developed, particularly using the BHV-1 glycoproteins, gI, gIII, and gIV. Purified BHV-1 glycoproteins appear not to be highly antigenic, as direct application of the purified proteins to nasal mucosa did not result in detectable antibody formation. However, using cholera toxin as an adjuvant, mucosal immunization resulted in the induction of antibodies, although the duration of immunity was not established (Israel et al., 1992, Virology, 188:256-264). Protection is disclosed using recombinant BHV-1 glycoproteins as immunogens, particularly in the presence of an adjuvant (Babiuk et al., U.S. Pat. No. 5,151,267 issued Sep. 29, 1992).
Diagnosis of BHV-1 infection in a herd is usually based on serum neutralization tests, but other diagnostic tests have been developed (for a review, see Straub et al., supra). Tests used to detect the presence of BHV-1 in semen include virus isolation techniques (Sheffy et al., 1973, Proc. 77th Annual Meeting of the U.S. Animal Health Assoc., 131-137; Darcel et al., 1977, Proc. 20th Annual Meeting Am. Assoc. Vet. lab. Diagnost., 209-214; Kahrs et al., 1977, Proc. 20th Annual Meeting Am. Assoc. Vet. Lab. Diagnost., 187-208), and the "Cornell Semen Test" (Schultz et al., 1982, Theriogenol. 17:115-123). The Cornell Semen Test is currently the most sensitive method for detecting viral infectious agents in bull semen. In this test, pooled semen samples are inoculated into susceptible calves or sheep which undergo subsequent serological testing. The method has several disadvantages in that it is not possible to recognize which specific sample(s) of the pool is contaminated; the costs of the animal isolation facilities are high; and seroconversion of the inoculated animals takes up to 3 weeks. Thus, the virus isolation techniques and serological testing are both labor and time intensive.
Recent advances in molecular biology have provided several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method, PCR (polymerase chain reaction, Cetus Corporation) involves the use of Taq Polymerase, known sequences as primers, and heating cycles which separate the replicating deoxyribonucleic acid (DNA) strands in exponentially amplifying a gene of interest. Other amplification methods currently under development include LCR (ligase chain reaction, Barnay, F., 1991, Proc. Natl. Acad. Sci. USA, 88:189-193) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified; enzyme QB replicase (Gene-Trak Systems) and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used to make a DNA template for exponential production of complementary RNA; and NASBA (nucleic acid sequence-based amplification, Cangene Corporation) which can be performed on RNA or DNA as the nucleic acid sequence to be amplified.
Nucleic acid probes that are capable of hybridization with specific gene sequences have been used successfully to detect specific pathogens in biological specimens at levels of sensitivity approaching 10.sup.3 -10.sup.4 organisms per specimen. PCR has been applied to the field of medical diagnostics and veterinary medicine. PCR-based detection systems for BHV-1 have recently been described by Vilcek (1993, J. Virol. Methods 41:245-248), and by Israel et al. (1992, Virology 188:256-264). Neither reference describes the application of a PCR-based assay for detecting BHV-1 in semen samples. Both of these assays describe visualization for the detection of the PCR products using agarose gel electrophoresis. The sensitivity of one of the assays is in the order of 10.sup.3 plaque forming units (PFU) per ml in spiked nasal secretions (Israel et al., supra).
Coupled with a method that increases the specificity of amplification of BHV-1 target DNA sequences, the nested set of primers of the present invention can increase the level of sensitivity in detecting BHV-1 in a clinical specimen. Use of these primers may allow direct detection without relying on prior culture, conventional serological testing, or agarose gel electrophoresis. The present invention is particularly suited for detecting the presence of BHV-1 in bull semen. By using the oligonucleotides of the present invention, BHV-1 can be detected at a level of 5.times.10.sup.3 TCID.sub.50 (50% tissue culture infectious dose) per 0.5 ml of semen, as compared to the current "gold standard" of 5.times.10.sup.3 to 2.5.times.10.sup.4 TCID.sub.50 in the Cornell Semen Test.